RNA splicing, the biochemical process leading to the removal of introns from precursor RNA, is an important step in the expression and regulation of genetic information. Nuclear pre-mRNA splicing requires ATP and occurs in a complex, dynamic structure called the spliceosome which is composed of small nuclear ribonucleoproteins. The long-term objective of this grant is to understand the mechanism of nuclear pre- mRNA splicing through biochemical and genetic analysis of the spliceosome and its components. A series of RNA rearrangements occur during the assembly and maturation of the spliceosome, and one of the driving forces for these rearrangement is the splicing ATPases. We have extensively characterized such an RNA-dependent ATPase, the yeast (Saccharomyces cerevisiae) Prp2 protein. Our studies suggest that Prp2 hydrolyzes ATP to activate the spliceosome for the first transesterification reaction. Recently, we detected several structural changes in the pre-mRNA and the U2 and U6 snRNAs as results of the spliceosome activation process medicated by PRP2. We hypothesize that the RNA nucleotides that are affected by PRP2 and ATP are likely to be involved in building up the catalytic center for splicing. In addition, a protein factor (HP) required for the transesterification reaction was also identified. Now we are in a unique position to describe and to investigate the significance of the RNA changes that are caused by a splicing ATPase, as well as the involvement of RNA and protein in catalyzing the splicing reaction. Furthermore, the characteristics of the Prp2 protein in spliceosome activation may serve as a model for other DExH proteins. There are three specific aims. (1) We will continue to investigate the RNA changes that occur during the activation of the spliceosome by Prp2 and ATP. We will test the hypothesis that the role of PRP2 is to form critical RNA interactions for splicing. (2) We will continue to purify the HP protein and to investigate the relationship among HP, oxygen in the phosphate backbone of U6, and metal ions. (3) Individual DEAH splicing ATPase apparently only binds to its "cognate" spliceosome. We plan to understand this specificity by searching for RNA and proteins that interact with PRP2 in the spliceosomal pocket.